REGULATION OF NA+-H+ EXCHANGE BY ATP DEPLETION AND CALMODULIN ANTAGONISM IN RENAL EPITHELIAL-CELLS

The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na+-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive Na-22+ uptake (P < 0.001; n = 12) and Na+-dependent intracellular pH (pH(i)) recovery in 2',7'-bis (carboxyethyl)-5(6)- carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na+-H+ exchange activity. The W-7-induced inhibition of Na+-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pH(i) dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/- 5% (P < 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P < 0.05; n = 6). ATP depletion significantly inhibited apical Na+-H+ exchange. These results suggest that an ATP-Ca2+-calmodulin-dependent process is involved in regulation of Na+-H+ exchange in LLC-PK1 cells. A Ca2+-calmodulin-dependent process activated the amiloride-sensitive basolateral Na+-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na+-H+ exchangers or regulatory proteins by a Ca2+-calmodulin-dependent protein kinase may mediate this differential regulation.